Mimetic insect allatostatin analogs for insect control

ABSTRACT

Novel pseudopeptide analogs of the insect allatostain neuropeptide family which possess biological activity mimicking that of the naturally ocurring neuropepetides are disclosed. By addition of a hydrophobic moiety to an active portion of the allatostatin peptides, analogs are produced which exhibit an overall amphipilic nature and which are capable of penetrating the insect cuticle while still retaining biological activity. Furthermore, by substituting sterically hindered amino acids or aromatic acids for any or all of the first, third or fifth amino acids residues of the allatostatin C-terminal pentapeptide, analogs may be produced which are resistant to degradation by insect peptidases while still retaining biological activity. The analogs may be used for insect control by disrupting critical reproductive and/or developmental processes normally regulated by allatostatins in insects.

This application is a division of Ser. No. 09/191,906 filed Nov. 13,1998, now U.S. Pat. No. 6,207,643.

BACKGROUND OF THE INVENTION

1. Field of Invention

This invention relates to mimetic pseudopeptide analogs of theallatostatin neuropeptide family, and the use of these analogs forinsect control.

2. Description of the Prior Art

The allatostatin family of insect neuropeptides inhibit the in vitrobiosynthesis of juvenile hormone (JH) by the corpora allata of thecockroaches Diploptera punctata Blattella germanica (L) and Periplanetaamericana (Belles et al., 1994, Reg. Peptides, 53:237-247; and Weaver etal., 1994, Comp. Biochem. Physiol., 107(c):107-127). In these pests, JHplays a significant role in development reproductive maturity, sexpheromone production and mating. Specifically, a reduction in endogenouslevels of JH is critical to development of the adult stage from thenymph in the cockroach, whereas oocyte growth and maturation in adultfemales show a dependency on the presence of JH (Bendena et al.,Allatostatins: Diversity in structure and function of an insectneuropeptide family. In: Beckwith et al., Eds., Neuropeptides inDevelopment and Aging, pp. 53-66, Annals NY Acad. Sci. 814: 53-66:1997).

Although the allatostatins can influence a number of physiologicalprocesses by virtue of their ability to modulate in vitro production ofJH, the native allatostatins have held little promise as insect controlagents. The major limitations of the allatostatins which have hamperedtheir use for insect control include their inability to penetrate theinsect cuticle, and their susceptibility to inactivation by peptidasesin the hemolymph and gut and/or bound to tissues within the insect(Bendena et al., ibid).

Earlier structure-activity studies have shown that the C-terminalpentapeptide Xaa₃-Xaa₁-Phe-Gly-Xaa₂-NH₂ (wherein Xaa₁ occurs as any ofAsn, Asp, Gly, Ser or Ala, Xaa₂ occurs as Leu or Ile, and Xaa₃ occurs asTyr or Phe) is shared by all members of the Diploptera allatostatins,and represents the ‘active core’ region or minimum sequence capable ofeliciting inhibition of JH production in vitro (Hayes et al., Peptides,15:1165-1171, 1994; Pratt et al., Biochem. Biophys. Res. Commun.,163:1243-1247, 1989; and Pratt et al., Proc. Natl. Acad. Sci. USA,88:2412-2416, 1991). The side chains of active core residues Phe, Leuand Tyr proved to be the most important for activity (Hayes et al.,ibid). Recent studies have elucidated the primary catabolic cleavagesites of the allatostatins following incubation with hemolymph enzymesand with membrane peptidases in crude membrane preparations. Hemolymphenzymes primarily cleave the peptides in the N-terminal region outsideof the pentapeptide core region and therefore, do not inactivate theallatostatins (Garside et al., Peptides, 18:17-25, 1997). However,membrane preparations of brain, gut and corpora allata cleaveallatostatins at the C-terminus between residues Gly-Leu, with secondarycleavage occuring between the residue block Xaa₃-Xaa₁ (Garside et al.,Gen. Comp. Endocrinol., 108:258-270, 1997). Both of these cleavagesdisrupt the active core sequence and lead to completely inactivefragments.

The contents of each of the above-mentioned publications areincorporated by reference herein.

SUMMARY OF THE INVENTION

We have discovered novel pseudopeptide analogs of the insectallatostatin neuropeptide family which possess biological activitymimicking that of the naturally occurring neuropeptides. By addition ofa hydrophobic moiety to an active portion of the allatostatin peptides,analogs are produced, which exhibit an overall amphiphilic nature andwhich are capable of penetrating the insect cuticle while stillretaining biological activity. Furthermore, by substituting stericallyhindered amino acids or aromatic acids for any or all of the second,third or fifth amino acid residues of the allatostatin C-terminalpentapeptide, analogs have been produced which are resistant todegradation by insect peptidases while still retaining biologicalactivity. The analogs may be used for insect control by disruptingcritical reproductive and/or developmental processes normally regulatedby allatostatins in insects.

In accordance with this discovery, it is an object of this invention toprovide novel compounds having biological activity mimicking that of thenaturally occurring allatostatin neuropeptides.

It is also an object of this invention to provide compounds which arebioactive mimics of allatostatin neuropeptides that are capable ofpenetrating the insect cuticle.

Another object is to provide compounds which are bioactive mimics ofallatostatin neuropeptides which are resistant to enzyme degradation.

Yet another object is to provide compounds which are bioactive mimics ofallatostatin neuropeptides and their use for controlling insectpopulations and which may be topically applied.

Other objects and advantages of the invention will become readilyapparent from the ensuing description.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the effect of time on the penetration of the9-fluoreneacetic acid pentapeptide analog of Example 1 through thecuticle of the cockroach.

FIGS. 2(a)-(c) show the structures of peptidase resistant allatostatinanalogs containing sterically hindered, restricted conformationcomponents: a) indane ring (Aic) analog 396-1, top left; b) cyclopropylring (Cpa) analog 397-2, top right; and c) cyclopropyl ring (Cpa) analogwith a hydrocinnamic acid (Hca) ‘cap’ replacement for Phe [AST(b)φ2],bottom center. Upward arrows denote cleavage sites of tissue-boundpeptidases in the cockroach. The large upward arrows with a cross overthem, indicate a peptidase cleavage site that is blocked by the presenceof the sterically hindered components and/or presence of thehydrocinnamic acid cap.

FIGS. 3(a) and (b) show the structures of peptidase resistantallatostatin analogs containing sterically hindered, restrictedconformation components: a) both an indane ring (Aic) and cyclopropylring (Cpa), and b) benzodiazepine (Bzd).

FIG. 4 shows the dose-response curve for inhibition of in vitro juvenilehormone JH biosynthesis in corpora allata of the cockroach Diplopterapunctata by allatostatin analog AST(b)φ2 in Example 2. Each point foranalog treatment represents the mean of 14-16 measurements +/− thestandard error of the mean.

DETAILED DESCRIPTION OF THE INVENTION

In the following description, the nomenclature used to define thepeptides and pseudopeptides is that specified by Schroder and Lubke[“The Peptides,” Academic Press (1965)] wherein, in accordance withconventional representation, the N-terminal appears to the left and theC-terminal to the right. Where the amino acid residue has isomericforms, it is the L-form of the amino acid that is represented hereinunless otherwise expressly indicated.

Allatostatin analogs capable of penetrating the insect cuticle areprepared by conjugating a hydrophobic moiety to a member of theallatostatin insect neuropeptide family, or a bioactive portion thereof,to render the compound amphiphilic. These compounds are of the generalformula I:

R-X₁-Phe-Gly-x₂-NH₂  (I)

wherein X₁ is Asn, Asp, Gly, Ser, or Ala, and X₂ is either Leu or Ile.The R moiety incorporates the hydrophobic functionality which iseffective to impart the amphiphilic nature to the molecule.

In a first preferred embodiment, the compound is a pseudopentapeptideanalog of the C-terminal allatostatin core region. In this embodiment, ahydrophobic carborane moiety is incorporated onto the N-terminus of theC-terminal pentapeptide, Xaa₃-X₁-Phe-Gly-X₂-NH₂ (Xaa₃ being Phe or Tyr),or the C-terminal tetrapeptide, X₁-Phe-Gly-X₂-NH₂. Specifically, o-, m-or p- carborane conjugated to a short chain alkanoyl-acyl group isconjugated to the N-terminal Tyr or Phe amino acid of the pentapeptide,or the N-terminal X₁ of the tetrapeptide. In a variation of thisembodiment, the aromatic ring of the Tyr or Phe amino acid (Xaa₃) may bereplaced by a carborane moiety, thereby forming carboranyl alanine ofthe formula:

Referring to formula I, in this first preferred embodiment R maytherefore be shown as:

Cb—(CH₂)_(n)—C(O)—X₃′  (III)

where Cb is a carborane, n is 1, 2 or 3, and X₃′ is Tyr, Phe, carboranylalanine or a bond. When X₃′ is a bond, n is preferably 2.

In a second preferred embodiment, the allatostatins or an activeC-terminal portion thereof containing at least the C-terminalpentapeptide Xaa₃-X₁-Phe-Gly-X₂-NH₂ (Xaa₃ being Phe or Tyr), aremodified at their N-terminus by addition of a hydrophobic moiety whichmay be an aromatic amine, aromatic acid, or aliphatic fatty acid. Avariety of hydrophobic aromatic amines and acids are suitable for useherein. Preferred acids include phenyl alkanoic, alkenoic or alkynoicacids such as 9-fluoreneacetic acid, 6-phenyl hexanoic acid or 9-phenylnonanoic acid, while preferred amines include phenyl alkanoic, alkenoicor alkynoic amines such as 4-phenyl butyl amine. Without being limitedthereto, examples of other suitable aromatic acids include2-biphenylenecarboxylic acid, 9-anthracenecarboxylic acid,1-fluorenecarboxylic acid, and 1-pyrenebutyric acid, while othersuitable aromatic amines include 1-aminoanthracene,6-amino-3,4-benzocoumarin, 2-amino-7-bromofluorene, 6-aminochrysene,3-aminofluoranthene, 9-aminophenanthrene, and 1-pyrenemethylamine, andsuitable aliphatic fatty acids include palmitic acid, caprylic acid,decanoic acid, lauric acid, and valeric acid. Optionally, thehydrophobic aromatic amines or acids or the aliphatic fatty acids mayfurther include the amino acid Arg conjugated thereto.

The analog of this second embodiment may be prepared from any member ofthe allatostatin family of neuropeptides. A variety of theseallatostatins have been previously described and are suitable for useherein. As mentioned, the allatostatin polypeptide to which thehydrophobic moiety is attached should include at least an activeC-terminal portion containing the C-terminal pentapeptideXaa₃-X₁-Phe-Gly-X₂-NH₂, and may encompass the entire native allatostatinpolypeptide. However, the polypeptide should not be so large as to losethe hydrophobic character introduced by the hydrophobic moiety. Thus,without being limited thereto, the size of the polypeptide (includingthe above-mentioned C-terminal pentapeptide) is usually less than orequal to about 20 amino acids, particularly less than or equal to about10 to 12 amino acids. As described in the first embodiment, the aromaticring of the Tyr or Phe amino acid (Xaa₃) may again be replaced by acarborane moiety (i.e. substituting carboranyl alanine for the Phe orTyr)..

The above-mentioned hydrophobic aromatic acids or amines or thealiphatic fatty acids may be conjugated to the allatostatin peptidedirectly or through an optional spacer. Use of the spacer is preferredhowever, to minimize any steric hindrance of the active polypeptideportion of the compound by the hydrophobic moiety and inhibition ofreceptor binding. The structure of the spacer will vary with theparticular hydrophobic group. Without being limited thereto, when thehydrophobic moiety is an aromatic acid or an aliphatic fatty acid,preferred spacers may be non-polar hydrocarbons having a free aminogroup and free carboxyl group, or relatively non-polar or unchargedα-amino acids, such as Ala, Ala-Ala dimer, or Gly. When using anaromatic amine as the hydrophobic moiety, preferred spacers arehydrocarbon diacids, such as succinic acid. Other specific spacers maybe readily determined by the practitioner skilled in the art.

In summary, in accordance with the structure shown in Formula (I), thestructure of R for the second embodiment may be shown as:

R₁-L_(m)-X₄-R₂-X₃-  (IV)

where R₁ is the above-mentioned hydrophobic moiety, L is the spacer, mis 0 or 1, X₄ is a bond or arg, and X₃ is Tyr, Phe or carboranylalanine. The group R₂ may be a bond, or an amino acid or polypeptidewhich is naturally contiguous to the C-terminal pentapeptideXaa₃-X₁-Phe-Gly-X₂-NH₂. Without being limited thereto, specific examplesof suitable R₂ moieties include:

-Leu-,

-Ala-Tyr-Ser-Tyr-Val-Ser-Glu-Tyr-Lys-Arg-Leu-Pro-Val- (SEQ ID No. 1),

-Ser-Lys-Met-,

-Asp-Gly-Arg-Met- (SEQ ID No. 2),

-Asp-Arg-Leu-,

-Ala-Arg-Pro-,

-Ala-Pro-Ser-Gly-Ala-Gln-Arg-Leu- (SEQ ID No. 3),

-Gly-Gly-Ser-Leu- (SEQ ID No. 4),

-Gly-Asp-Gly-Arg-Leu- (SEQ ID No. 5),

-Pro-Val-Asn-Ser-Gly-Arg-Ser-Ser-Gly-Ser-Arg- (SEQ ID No. 6),

-Tyr-Pro-Gln-Glu-His-Arg- (SEQ ID No. 7), and

-Pro-.

In a third embodiment for the preparation of allatostatin analogscapable of penetrating the insect cuticle, the aromatic ring of the Tyror Phe amino acid (Xaa₃) is replaced by a carborane moiety. While thismodification is the same as described in the first and secondembodiments (substituting carboranyl alanine for the Phe or Tyr), thisembodiment does not require the use of additional hydrophobic groups asdescribed in the earlier embodiments. The resultant pseudopentapeptideanalog may include an optional amino acid, Arg, conjugated to the freeamine group of the carboranyl alanine.

In summary, referring to Formula 1, in this third embodiment thestructure of R may be shown as:

R₃-carboranyl alanine-

wherein R₃ is H or Arg.

In accordance with a further embodiment, the invention also encompassesthe preparation of allatostatin analogs which are resistant todegradation by insect peptidases while still retaining biologicalactivity. These analogs may be prepared by substituting selectedsterically hindered amino acids and/or aromatic acids for the second,third and/or fifth amino acids of the allatostatins or active C-terminalportions thereof containing the C-terminal pentapeptideXaa₃-X₁-Phe-Gly-X₂-NH₂ (Xaa₃, X₁, and X₂ being as described above).

The substituted amino acids or aromatic acids incorporated into thepentapeptide must possess sufficient bulk as to sterically hindercleavage of the analog by peptidases in the target insect, yetsubstantially retain the secondary structure and active conformation ofthe native allatostatin pentapeptide necessary for biological activity.In this embodiment, the Phe of the native C-terminal pentapeptide isreplaced with 2-amino-indane-carboxylic acid (Aic), and/or the Gly isreplaced with either cyclopropyl alanine (Cpa) or cyclobutyl alanine(Cba). Alternatively, both the Phe and Gly may be replaced with1,4-benzodiazepine (Bzd). These substitutions may be incorporated intothe complete allatostatins or into active C-terminal portions thereofwhich contain the C-terminal pentapeptide.

All of the Bzd, Cpa, and Aic containing analogs retain significantbiological activity and increased resistance to peptidase degradation.The Aic containing analogs in particular exhibit biological activitycomparable to the native allatostatin C-terminal pentapeptide. Each ofthe Aic, Cpa, and one of the diastereomers of the Bzd containing analogsexhibit inhibition of JH biosynthesis in the cockroach in thephysiological (μM or lower) range. Interestingly, rather than inhibitingJH biosynthesis, the other diastereomer of the Bzd containing analogactually stimulates JH biosynthesis in the cockroach. Despite theseapparent differences, all of the analogs are effective for insectcontrol as described hereinbelow. Although these analogs exhibitresistance to degradation by hemolymph peptidases as well as membranebound peptidases at their primary cleavage site, between Gly and Leu-NH₂in the native pentapeptide, they are still susceptible to degradation bythe membrane bound peptidases at their secondary cleavage site near theN-terminal end of the pentapeptide.

In contrast to the allatostatin pentapeptide analogs incorporatingsterically hindered amino acids at the second or third amino acids,analogs prepared by substitution of hydrocinnamic acid (Hca) orhydroxycinnamic acid (Hhca or hydroxyphenyl propionic acid) for theN-terminal Xaa₃ amino acid (Tyr or Phe) of the C-terminal allatostatinpentapeptide, exhibit significantly increased resistance to degradationby membrane bound peptidases near the N-terminus of the molecule. Theseanalogs also retain significant biological activity. The Hca or Hhcareplace the N terminal Tyr or Phe, capping the peptide and effectivelyremoving the N-terminal cleavage sites of the membrane bound peptidaseswithin the native pentapeptide, and also blocking the N-terminal primarycleavage sites of the hemolymph enzymes.

While the above-described Cpa, Aic, Bzd, and Hca (or Hhca) substitutionsmay be incorporated into the analog individually, in the preferredembodiment, optimal resistance to insect peptidases is achieved bysubstitution of each of the second, third and fifth amino acids asdescribed. Thus, in the preferred embodiment, the analogs willincorporate both the substitution of an Hca or Hhca for Xaa₃, andsubstitution of the Phe or Gly with Aic or Cpa, respectively, orsubstituting both Phe and Gly with Aic-Cpa or with Bzd.

In review, the peptidase-resistant allatostatin analogs of thisinvention may be generally shown by the formula:

R′ -X₁-X_(a)-X_(b)-X₂-NH₂  (V)

wherein X₁ and X₂ are as described above in formula I. R′ may be Phe,Tyr, a hydrocinnamyl group, a hydroxyhydrocinnamic acyl group, or apolypeptide group which may be all or a portion of an allatostatinneuropeptide which is naturally contiguous to the C terminaltetrapeptide X₁-Phe-Gly-X₂-NH₂. X_(a)-X_(b) may be any of Phe-Caa,Aic-Gly, Aic-Caa, or Bzd, wherein Caa is a cycloalkyl alanine selectedfrom cylcopropyl alanine or cyclobutyl alanine, Aic is a2-amino-indane-2-carboxyl group, and Bzd is a 1,4-benzodiazepine group.Detailed structures of peptidase resistant analogs representative ofthis embodiment are shown in FIGS. 2 and 3. The peptidase resistantanalogs of this invention are also described in Nachman et al. (1997,Active Conformation and Peptidase Resistance of ConformationallyRestricted Analogues of the Insect Allatostatin Neuropeptide Family, IN:Kawashima and Kikuyama, Advances in Comparative Endocrinology, Bologna,Italy: Monduzzi Editore, pp. 1353-1359), Garside et al. (1997,Catabolism of Insect Neuropeptides: Allatostatin as Models forPeptidomimetic Design, IN: Kawashima and Kikuyama, Advances inComparative Endocrinology, Bologna, Italy: Monduzzi Editore, pp.169-173), and (1998, Bioorganic & Medicinal Chemistry, 6:1379-1388) thecontents of each of which are incorporated by reference herein.

Although the preparation and use of analogs capable of penetrating theinsect cuticle and analogs resistant to peptidase degradation may bepracticed separately, it is also understood that analogs may be preparedwhich incorporate the modifications of each embodiment. Thus in aparticularly preferred embodiment, a hydrophobic moiety is conjugated tothe allatostatin peptide or a bioactive portion thereof, while one orboth of Aic or Cpa are substituted for the Phe or Gly, respectively, orBzd is substituted for both Phe and Gly, within the C-terminalpentapeptide core.

The analogs of this last embodiment may be shown by the formula:

R₁-X₁-X_(a)-X_(b)-X₂-NH₂  (VI)

wherein R, X₁, and X₂ are as described in formula I, and X_(a)-X_(b) areas described in formula V hereinabove.

The peptides and pseudopeptide analogs of this invention may besynthesized by any suitable method, such as exclusively solid-phasetechniques, partial solid-phase techniques, fragment condensation, orclassical solution addition. The groups or amino acids of the compoundsof the invention, including the R groups, are typically joined toadjacent groups through amide linkages. The peptides may also besynthesized by recently developed recombinant DNA techniques which maybe utilized for large-scale use.

Synthesis by the use of recombinant DNA techniques, for the purpose ofthis application, should be understood to include the suitableemployment of structural genes coding for the sequence as specifiedhereinafter. The synthetic peptides may also be obtained by transforminga microorganism using an expression vector including a promoter oroperator, or both, together with such structural genes and causing suchtransformed microorganisms to express the peptide.

As stated above, the compounds of formulas I-VI can be synthesized bymethods well known to those skilled in the art of peptide synthesis,e.g., solution phase synthesis [see Finn and Hoffman, In “Proteins,”Vol. 2, 3rd Ed., H. Neurath and R. L. Hill (eds.), Academic Press, NewYork, pp. 105-253 (1976)], or solid phase synthesis [see Barany andMerrifield, In “The Peptides,” Vol. 2, E. Gross and J. Meienhofer(eds.), Academic Press, New York, pp. 3-284 (1979)], or stepwise solidphase synthesis as reported by Merrifield [J. Am. Chem. Soc. 85:2149-2154 (1963)], the contents of each of which are incorporated hereinby reference. In the preferred embodiment, the allatostatin polypeptidesand the analogs may be synthesized using the same solid phase techniquesdescribed by Nachman et al. (1995, Reg. Peptides, 57:359-370) orChristensen et al. (1991, Proc. Natl. Acad. Sci., USA, 88:4971-4975),the contents of each of which are incorporated by reference herein. Cbeor 2-o-carboranylpropanoic acid may be synthesized according topreviously described procedures (Radel and Kahl, 1993, Amino Acids,5:170, the contents of which are incorporated by reference herein). Thehydrophobic moieties may be incorporated into the analogs using thetechniques described in Nachman et al. for the preparation of pyrokininanalogs (U.S. Pat. No. 5,795,857, issued Aug. 18, 1998, the contents ofwhich are incorporated by reference herein).

The pseudopeptide analogs of formulas I-VI mimic the biological activityof the naturally occurring allatostatin neuropeptides and havedemonstrated the ability to disrupt or alter physiological processesfollowing application to an insect. In a preferred embodiment, with oneexception, the pseudopeptide analogs of each of the embodiments areeffective for inhibiting juvenile hormone production in cockroaches,thereby causing infertility in adults. Furthermore, inhibition ofjuvenile hormone production in immature insects causes precocious adultdevelopment resulting in sexual dysfunction. As noted above, rather thaninhibiting juvenile hormone biosynthesis, one of the diastereomers ofthe Bzd containing analogs stimulates production of the hormone incockroaches. Application of these analogs to immature insects mayprevent or inhibit their development to adults. The compounds appear tobe particularly suited for control of insects of the order Dictyopteraand Orthoptera, especially cockroaches and locusts.

As a practical matter it is anticipated that compositions of thepseudopeptide analogs would be prepared by formulating the compoundswith an agriculturally acceptable inert carrier, particularly a solventsuitable for topical applications. Although a variety of solvents may beused, water is preferred. The compounds may also be formulated withsolid inert carriers such as talc, clay or vermiculite, or incorporatedinto conventional controlled release microparticles or microcapsules. Inaddition, the analogs may be optionally formulated in combination withconventional insect attractants, baits, or other chemical or biologicalinsecticides.

The allatostatin analogs of this invention may be applied directly tothe target insects (i.e., larvae, pupae and/or adults), or to the locusof the insects. Because the compounds incorporating hydrophobic moietieswill penetrate the insect cuticle, they are preferably administeredtopically, such as by direct spraying on the insect or a substrate whichis likely to be contacted by the insect. Alternatively, the compoundsmay also be administered either subcutaneously, percutaneously, ororally. When they are to be ingested, they should be applied with theircarrier to the insect diet. The compounds are administered in an amounteffective to induce the desired response as determined by routinetesting. For example, where the desired effect is the inhibition ofjuvenile hormone biosynthesis, an “effective amount” is defined to meanthose quantities which will result in a significant decrease in juvenilehormone production in a test group as compared to an untreated control.Similarly, where the ultimate response is pest mortality, an “effectiveamount” is defined as those quantities which will result in asignificant mortality rate in a test group as compared to an untreatedcontrol. The actual effective amount will of course vary with thespecific compound, the target insect and its stage of development, theapplication technique, the desired effect, and the duration of theeffect, and may be readily determined by the practitioner skilled in theart. When determining effective amounts, it is understood that theseanalogs need not be as potent as the natural allatostatin peptide todisrupt physiological processes such as juvenile hormone biosynthesis,because their effects can be exerted over a considerable time, as aconsequence of their resistance to peptidase degradation. Without beinglimited thereto, it is envisioned that when administering the analogs byingestion, effective inhibition of juvenile hormone biosynthesis may beachieved using concentrations of between about 100-500 picomoles/insect.When the compounds are to be topically applied, the effective amountsmay be significantly higher.

It is envisioned that the compounds encompassed herein may be effectivefor controlling a variety of insects. Without being limited thereto,pests of particular interest are agronomically or commercially importantinsects, especially cockroaches and locusts.

The following examples are intended only to further illustrate theinvention and are not intended to limit the scope of the invention whichis defined by the claims.

EXAMPLE 1 Materials and Methods

Cuticle Preparation

Adults of the American cockroach were obtained from colonies at ourfacility (CMAVE). Animals, anesthetized by submersion in H₂O for 30 min,were pinned ventral side up in a wax dissecting dish flooded with water.Lateral incisions along the margins of the abdominal sternites were madebetween segments 1-7. The epidermal layers including the cuticle andepidermal cells were lifted up and associated tissue was cleared usingforceps prior to removal. The epidermal tissue was placed cell side upon a microscope slide and the cells were scraped from the cuticle usinga glass cover slip. To further clean the cuticle of cellular debris thetissue was floated, cell side down, in a beaker containing H₂O andsubjected to sonication in a water bath at 30° C. for 30 min. Thecuticle strips were then washed 3× in clean water. Pieces of cuticle,ca. 0.4 cm², composed of individual sternites without associatedintersegmental membranes were then dissected away from remainingcuticle.

Incubation of Cuticle

Incubation of cuticle was accomplished using the wells of ELISA plates(Corning, 96 well Easy Wash). Prior to use the wells of the plates wereblocked to minimize adsorption of analogs to the wells by filling with a1% gelatin in 10 mM sodium phosphate buffer containing 150 mM NaCl (pH7.25) (PBS) and incubating at 35° C. for 1.5 h. After blocking, theplates were washed with PBS-Tween and followed by three washes with H₂0.The wells of the plates were filled with 300 μl of H₂0 and pieces ofcuticle were floated, cell side down, in the wells. The 9-fluoreneaceticacid pentapeptide analog (0.5 nmol) was applied to the center of thecuticle pieces in a 0.5 μl drop of H₂0 using a Hamilton 10 μl syringefitted with a fused silica needle (0.17 mm OD) and held in a Brinkmannmicromanipulator. All applications and transfers were made using amicroscope and the preparations were observed for 5 min afterapplication of the analogs to insure that drops did not slide off thecuticle. Data from wells in which the drops slid off the cuticle werenot considered for analysis. Lids were applied to the plates after thedrops of water containing the analogs dried (ca. 5 min afterapplication) and the plates were placed on an orbital shaker operated at80 rpm. At this speed no water contacted the upper surface of thecuticle. One h after application of the analogs we carefully transferredthe cuticle to new wells containing 300 μl of H₂0 and incubation wascontinued. Cuticle was subsequently transferred to new wells atintervals after application of the analog. After incubation, 100 pmol ofinternal standard were added to the wells. The contents of the wellswere mixed by pumping the contents in and out of a micro pipette severaltimes and then transferred to a 1.5 ml microfuge tube. The wells wereextracted two additional times by addition of 350 μl of water and theextracts combined prior to concentration to apparent dryness using aSpeedVac concentrator.

Dried samples were reconstituted in 35% acetonitrile (MeCN) (Burdick andJackson) prior to analysis. Reversed phase liquid chromatographicanalysis (RPLC) was accomplished using a LDC Biochrome quaternarygradient pump and LDC Spectro Monitor 3200 UV detector set at 210 nm andinterfaced to a Nelson Analytical 3000 data acquisition and analysissystem. A Macrosphere 300 C18 reversed phase column (250 mm×2.1 mm id, 5μm, Alltech) was used for all separations. Solvents used for allseparations were H₂O and MeCN each containing 0.1% TFA as the ion pairreagent. Samples were injected onto the column using a Rheodyne 7125injector (100 μl loop) in 35% MeCN. The column was eluted after a 5 minequilibration period using a linear gradient from 35%-75% MeCN over 90min at a flow rate of 0.25 ml/min. Analysis of equimolar amounts ofanalogs indicated that the compounds had different detector responseswhen analyzed with the UV detector set at 210 nm. Therefore, all valueswere corrected to reflect the differential detector response for theanalogs. Data acquired from analyses were reduced and analyzed usingNCSS 97 software using regression analysis and T-Tests.

Radiochemical Assay

Rates of JH release were determined by the in vitro radiochemical methodof Feyereisen and Tobe (Anal. Biochem., 111:372-375, 1981) and asmodified by Tobe and Clarke (Tobe and Clarke, Insect Biochem.,15:175-179, 1985). The incorporation of L[¹⁴C-S-methyl]-methionine (50μM, specific radioactivity 1.48-2.03 GBq/mmol from New England Nuclearor Amersham) into JH III at its penultimate step of biosynthesis by CAincubated in 50 μl TC 199: (GIBCO, 1.3 mM Ca²⁺, 2% Ficoll,methionine-free) was used to quantify JH release. Animals wereanaesthetized on ice prior to dissection. Corpora allata were dissecteddirectly into non-radioactive medium (only animals with oviposited basaloocytes were used for assay). Samples were dried with nitrogen andresuspended in 10 μl 1N HCl. Radioactive medium was added andneutralized with 10 μl NaOH. Individual CA were transferred fromnon-radioactive medium to radioactive medium and were incubated for 3 h.The amount of inhibition was expressed as the percent reduction from theuntreated rate: [1− (treated rate/untreated rate)] ×100%. Each valuerepresents replicate incubations of a minimum of 8 tests.

Controls

All experiments were run with appropriate controls (Dip-AST5 incubatedfor 120 min with no membrane preparations added). If the identicalamount of AST was incubated under the same conditions with saline alone,the size of the HPLC-detected peak (U.V. 214 nm) remained constant overa 120 min period. The addition of 200 μl of 30% aqueous TFA completelyinactivated the enzymes in the membrane preparations and in intact CA.

Results

Analysis of samples obtained when using cockroach cuticle indicated thatthe analog penetrated the cuticle effectively (FIG. 1). The total amountof each of the analogs recovered over time increased in a logarithmicfashion (r=0.988, n=7/sample interval) over the test period. Althoughthe amount released per hour declined significantly during the first sixhours after application the release rate stabilized after six hours toabout 1.5 pmol/h. The logarithmic rates of release of the three analogsthrough the cuticle of the cockroach demonstrated that the cuticle wasacting as a slow release matrix for the analog. The initial rapid ratesof release indicated that the cuticle at the region of application wassaturated with pseudopeptide and that the analog was penetrating at itsmaximal rate. As time progressed, analog was absorbed and distributedmore evenly in the cuticle surrounding the point of application.Therefore, equilibrium was established throughout the cuticle so thatthe rate of release was reduced and declined slowly in a linear fashion.

The activity of the 9-fluoreneacetic acid pentapeptide analog (analog525) was 2.6×10⁻⁷ M (ED₅₀ or EC₅₀). In another assay, the activity ofthe carborane containing analog, carboranyl propanoicacid-Ser-Phe-Gly-Leu-NH₂, was 6.442×10⁻¹⁰ M (ED₅₀)

EXAMPLE 2 Materials and Methods

Allatostatin Analog Synthesis

The allatostatin analogs 396-1 (Ala-Arg-Pro-Tyr-Asn-Aic-Gly-Leu-NH₂,Aic=2-amino-indane-2-carboxyl-) and 397-2(Ala-Arg-Pro-Tyr-Asn-Phe-Cpa-Leu-NH₂, Cpa=cyclopropylAla) weresynthesized as previously described Nachman et al. (1997, 1998, ibid).The pseudopeptide allatostatin analog AST(b)φ2 (Hca-Asn-Phe-Cpa-Leu-NH₂,Hca=Hydrocinnamyl- and Cpa=cyclopropylAla-) was synthesized utilizingFMOC protection chemistry on MBHA resin (0.97 meq/gm substitution;Advanced Chemtech, Louisville, Ky.). Coupling reagents used for themajority of the amino acid condensations were 1 eq. of 1,3-diisopropylcarbodiimide/1-hydroxy-7-azabenzotriazole (HOAt) mixture indimethylsulfoxide for 2 h according to previously described procedures(Nachman et al., Peptides, 18:53-57, 1997). However, for coupling of theCpa residue and the residue immediately following Cpa to thepeptide-resin complex the reagent used was 1 eq.[O-(7-azabenzotiazol-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate] (HATU)(PerSeptive Biosystems, Marlborough, Mass.)with 2 eq. N,N-diisopropylethylamine in dimethylsulfoxide for 4 h.Removal of the N-terminal FMOC group from the Cpa was accomplished with20% piperidine in dichloromethane for 1 h rather than the 30 min usedfor the other residues. The peptide was cleaved from the resin with HFaccording to previously described conditions (Nachman et al., PeptideRes., 2:171-177, 1989). The crude product was purified on a Waters C18Sep Pak cartridge followed by a Delta Pak C18 reverse phase column on aWaters model 510 HPLC controlled with a Millenium 2010 chromatographymanager system (Waters, Milford, Mass.) with detection at 214 nm atambient temperature. Solvent A 0.1% aqueous trifluoroacetic acid (TFA);solvent B =80% aqueous acetonitrile containing, 0.1% TFA. Conditions:Initial solvent consisting of 20% B was followed by Waters linearprogram 6—100% B over 40 min; flow rate 2 ml/min. Retention timeHca-Asn-Phe-Cpa-Leu-NH₂): 15.0 min. The pure pseudopeptide analog wasanalyzed and quantified by amino acid analysis under previouslydescribed conditions, revealing the following analysis: F(1.0), L(1.0)and N(0.9). A fast atom bombardment (FAB) mass spectrum was obtained byadding 10 g of pseudopeptide sample to glycerol (1.5 μl) on a copperprobe, followed by bombardment with 8 kV Xe atoms on a Kratos MS-50 massspectrometer (Kratos, Manchester, UK). The structural identity and ameasure of the purity of the pseudopeptide was confirmed by the presenceof the following molecular ion (MH+): Hca-Asn-Phe-Cpa-Leu-NH₂, 607.4(Calc. MH⁺: 607.32).

Animals

D. punctata females mate almost immediately following emergence.Therefore, newly emerged, mated females were transferred each day fromthe stock culture to an incubator and kept at 27±1° C. The relativehumidity was approximately 50% with a 12 hour light:dark cycle. Insectswere reared on Purina Lab Chow and water. Mating was confirmed by thepresence of a spermatophore. Basal oocyte length was also measured (day5=1.44-1.68 mm). Only day 5 mated females were utilized for thedegradation studies. Day 7 mated females (only with oviposited basaloocytes) were the source of corpora allata for analysis of JHbiosynthesis.

Solid Phase Extraction

Preparation

Pasteur pipettes were used to make C₁₈ reversed-phase columns. A glassbead was placed in the bottom of each Pasteur pipette and approximately200 mg 125 Å C₁₈ bulk packing material was added. Glass wool was placedover the top of the packing material to hold it in place.

Procedure

Each column was washed with 1.5 ml of 0.1% BSA in 0.1% aqueous TFA,followed by 1.5 ml of 40% acetonitrile in 0.1% TFA and finally, 1.5 mlof 0.1% aqueous TFA. Samples were diluted in 0.2 ml 0.1% aqueous TFA andapplied to column. Eluant was reapplied to the column 2×. The column waswashed with 1.5 ml 0.1% aqueous TFA followed by 17% acetonitrile in 0.1%aqueous TFA. The allatostatins and their metabolites were eluted with0.5 ml 40% acetonitrile in 0.1% aqueous TFA into 12 mm×75 mm culturetubes and were dried with a Speed-Vac.

Protein Assay

Protein content of membrane preparations was determined by the method ofBradford (Bradford, Anal. Biochem., 72:248-254, 1976) using BSA asstandard. The average of three separate determinations was used.

Allatostatin Degradation Assay

Tissue Collection

Day 5 mated female D. punctata were anaesthetized on ice beforedissection. Following dissection, tissues were stored in saline (0.9 %NaCl pH 7.0-7.2 or cockroach saline; NaCl 150 μM, KCl 12 μM, CaCl₂.6 H₂O10 μM, MgCl₂ .6 H₂O 3 μM, glucose 40 μM, HEPES 10 μM at pH 7.2-7.4) onice. Tissues were cleaned of fat body and trachea and guts were cleaned.Midgut peritrophic membrane was removed, midguts cut open, the gutsgently pulled through forceps and then washed 3X. Tissues were stored at−70° C. until sufficient material was collected.

Membrane Preparation

Tissues were pooled and homogenized in saline on ice in microcentrifugetubes (2.5 ml) for 2 min with an Omni hand held homogenizer. Homogenateswere centrifuged at 1000 g for ten min at 4° C. to remove cellulardebris. The. pellet was discarded. Supernatant was subsequentlycentrifuged at 30,000 g for 30 min. Pellet (crude membrane preparation)was washed three times in homogenization buffer and resuspended insaline using the

Omni homogenizer.

Hemolymph Preparation

Hemolymph was collected following the method of King and Tobe (InsectBiochem., 18:793-805, 1988). Hemolymph was diluted 100× with saline.

Assay

Crude membrane preparation or hemolymph was aliquoted into 1.5 mlmicrocentrifuge tubes for in vitro assay. Dip-AST5 (6 μM) or analog wasadded to each assay tube from a 1 mM stock solution. Total incubationvolume was 500 μl. Tubes were placed on a shaker at room temp for theduration of the incubation time. Incubations were terminated by theaddition of 200 μl 30% aqueous TFA. Samples were centrifuged at 10,000 gand the supernatant was applied to a C₁₈ solid phase extraction column.None of the potential, synthetic Dip-AST5 metabolites are lost(unpublished data). The 17-40% acetonitrile fraction was concentratedwith a Speed-Vac apparatus and held at −70° C. until HPLC analysis.Samples were diluted in 0.5 ml 0.1% aqueous TFA, filtered through 0.2 μmmicro-spin filters and injected onto the HPLC column. Separation ofallatostatins and their catabolites, or analogs and their catabolites,was performed using reversed-phase HPLC (see HPLC methods). Whenavailable, retention times of synthetic metabolites were used as areference to identify endogenous metabolites. Dip-AST5 fragments weresynthesized by the Institute for Biosciences and Technology, Dept. ofEntomology, Texas A & M University. Dip-ASTs were synthesized by theCore Facility of Insect Biotech Canada (Department of Biochemistry,Queen's University) or obtained from Sigma Chemical Co., St. Louis, Mo.63178 USA. The identity of fragments resulting from peptidasedegradation was also confirmed by FAB mass spectrometry by addingsamples in a glycerol medium (1.5 μl) onto a copper probe, followed bybombardment with 8 kV Xe atoms on a Kratos MS-50 mass spectrometer(Kratos, Manchester, UK).

HPLC

Degradation experiments were analyzed by RP-HPLC using a 220×4.6 mmBrownlee Phenyl column (5 μm) on a Spectra-Physics chromatography systemwith a Spectra-Physics Chromjet integrator and a Spectra-Physics 8490detector. Following a 5 min wash with 15% acetonitrile in 0.1% aqueousTFA, a linear gradient of acetonitrile (15-37.5% in 50 min; 37.5-65% in5 min) at a flow rate of 0.5 ml/min was used to elute peptides andanalogs. Fractions (0.5 ml) were collected each minute during thegradient run.

Radiochemical Assay

Radiochemical assays were conducted as described in Example 1.

Controls

Controls were prepared and conducted as described in Example 1.

Results

In analog 396-1 (Ala-Arg-Pro-Tyr-Asn-Aic-Gly-Leu-NH₂), the Phe residuewas replaced with an indane ring system (abbreviatedAic=2-Amino-indane-2-carboxyl-; see FIG. 1) which preserved the presenceof the side chain phenyl ring, a structural feature critical forbiological activity (Hayes et al., ibid). In a second analog (397-2:Ala-Arg-Pro-Tyr-Asn-Phe-Cpa-Leu-NH₂), the Gly residue was replaced witha cyclopropyl ring system (abbreviated Cpa=cyclopropylAla-, see FIG. 2).Molecular dynamics analyses, incorporating distance and angleconstraints obtained from NMR spectroscopy, were conducted separatelyfor each of the analogs of the series.. All demonstrated a preferencefor a very similar low energy turn over the four C-terminal residues.

Both allatostatin analogs 396-1 and 397-2 retained significantbioactivity (Table 2); specifically, 396-1 has an IC₅₀ 2.12 nM whichcompares favorably to Dip-AST5 which has an IC₅=3.1 nM. Patterns ofpeptidase hydrolysis by hemolymph enzymes for analogs 396-1 and 397-2indicate that the analogs were targeted at peptide bonds outside of theactive core region. The analogs were cleaved near the N-terminus,initially at Arg-Pro, followed by cleavage at Pro-Tyr. These sites ofcleavage are directly comparable to the sites of cleavage in the naturalpeptide Dip-AST5 by soluble hemolymph enzymes (Arg-Leu and Leu-Tyr)(Garside et al., Peptides, 18:17-25, 1997).

Surprisingly, these analogs showed little resistance to degradation byenzymes in crude membrane preparations. Whereas the modificationssuccessfully prevented truncation of the C-terminal Leu-NH₂, bothanalogs were still degraded at secondary cleavage sites near theN-terminus, similar to those observed following incubation ofallatostatins with hemolymph enzymes. Membrane-bound (brain and midgut)peptidases cleaved 396-1 first at Ala-Arg, followed by cleavage atPro-Tyr and then at Asn-Aic yielding the C-terminal tripeptide(Aic-Gly-Leu-NH₂). Analog 397-2 was initially cleaved at Arg-Profollowed by cleavage at Tyr-Asn. Interestingly, we found no evidence ofaminopeptidase removal of the N-terminal Ala in analog 397-2.

Since cleavage of analogs 396-1 and 397-2 occurred near the N-terminus,we synthesized analog AST(b)φ2 (FIG. 1), which retained the cyclopropylring system of 397-2 at the C-terminus and also incorporated anunnatural, non-amino acid group, hydrocinnamic acid, at the N-terminus.The hydrocinnamic acid group replaces the critical N-terminal Tyr,thereby effectively removing both cleavage sites suggested from theincubation of Dip-ASTs with hemolymph enzymes. This also effectivelyremoved the sites targeted by membrane-bound enzymes in analog 397-2.The hydrocinnamic acid moiety lacks the phenolic OH group and N-terminalamino group of the Tyr residue. This analog exhibited extreme resistanceto degradation by enzymes in hemolymph and in crude membranepreparations of brain and midgut (Table 1). Assay of AST(b)φ2 forinhibition of JH biosynthesis indicates that it does retain significantbiological activity (FIG. 4).

Discussion

Studies on the structure-activity relationships of the allatostatinsindicate that the C-terminal pentapeptideTyr/Phe-Xaa-Phe-Gly-Leu/Ile-NH₂ represents the core sequence requiredfor functional allatostatin activity in vitro (Duve et al., Proc. Natl.Acad. Sci. USA, 90:2456-2460, 1993; Hayes et al. ibid; Pratt et al.,1989, ibid; Pratt et al., 1991, ibid). In terms of the inhibition of JHbiosynthesis, the amino acid side chain of Leu was the most importantside chain, followed by Phe and Tyr, all of which are located in theC-terminal core sequence. Recent studies have elucidated the primarycatabolic cleavage sites of allatostatins following incubation witheither soluble enzymes in the hemolymph or with membrane peptidases incrude membrane preparations (Garside et al., Peptides, 18:17-25, 1997;

and Garside et al., Gen. Comp. Endocrinol., 108:258-270, 1997).Hemolymph enzymes cleave Dip-AST5 primarily near the N-terminus atArg-Leu, to yield the C-terminal hexapeptide. This hexapeptide issubsequently cleaved at Leu-Tyr to yield the C-terminal pentapeptide(Garside et al., Peptides, 18:17-25, 1997). Interestingly, thesecleavages do not inactivate Dip-AST5 because they do not target siteswithin the core sequence of the allatostatins. However, the potency ofthese two catabolites is significantly reduced (Stay et al.,Allatostatins, neuropeptide inhibitors of juvenile hormone biosynthesisin brain and corpora allata of the cockroach Diploptera punctata, In:Menn et al., Eds., Insect Neuropeptides, Washington DC, AmericanChemical Society, 164-176, 1991). For example, the activity of theC-terminal pentapeptide is reduced about 1000-fold (Bendena et al.,ibid). Catabolic enzymes in crude membrane preparations of brain, gutand corpora allata cleave allatostatins primarily in the core C-terminalregion at Gly-Leu-NH₂, with a secondary cleavage site between Tyr-Xaa.Cleavage at the primary site completely inactivates the allatostatin interms of the inhibition of JH biosynthesis.

Analogs 396-1 and 397-2 were constructed with modifications in theC-terminal ‘active core’ region of the allatostatins to blockdegradative cleavage by membrane-bound enzymes. Surprisingly, theseanalogs showed little resistance to cleavage by membrane-bound enzymes.Nevertheless, the two analogs did block the membrane-bound degradationthat would occur at the primary cleavage site within the C-terminalactive core pentapeptide region, between Gly-Leu-NH₂ in native peptide.The observed cleavage pattern of 396-1 by membrane-bound (brain andmidgut) peptidases is consistent with aminopeptidase removal of Alafollowed by two consecutive dipeptidyl aminopeptidase-like (DAP)cleavages. In analog 397-2, the two observed cleavages are alsoconsistent with DAP-like removal of successive N-terminal dipeptides.Alternatively, the second cleavage observed with 397-2 (Tyr-Asn) may becatalyzed by a chymotrypsin-like enzyme, because it occurs on thecarboxyl side of an aromatic residue.

This N-terminal cleavage pattern observed with analogs 396-land 397-2provided the impetus for the design of analog AST(b)φ2, incorporatingthe cyclopropyl ring system from 397-2 and a hydrocinnamic acid group, amimic of the Tyr residue, to ‘cap’ the N-terminus. This analogeffectively removed cleavage sites suggested from incubation of Dip-ASTSwith hemolymph enzymes, and those sites targeted by membrane-boundenzymes in native allatostatins and in analog 397-2. The observedresults indicate that AST(b)φ2 is the first mimetic analog of an insectneuropeptide with resistance to both hemolymph and tissue-boundpeptidases. This analog demonstrated 8-fold and 600-fold longerhalf-lives than the native neuropeptide Dip-AST5 following exposure tohemolymph and brain peptidases, respectively, and proved to becompletely resistant to midgut peptidases.

It is understood that the foregoing detailed description is given merelyby way of illustration and that modifications and variations may be madetherein without departing from the spirit and scope of the invention.

14 1 13 PRT Diploptera punctata 1 Ala Tyr Ser Tyr Val Ser Glu Tyr LysArg Leu Pro Val 1 5 10 2 4 PRT Diploptera punctata 2 Asp Gly Arg Met 1 38 PRT Diploptera punctata 3 Ala Pro Ser Gly Ala Gln Arg Leu 1 5 4 4 PRTDiploptera punctata 4 Gly Gly Ser Leu 1 5 5 PRT Diploptera punctata 5Gly Asp Gly Arg Leu 1 5 6 11 PRT Diploptera punctata 6 Pro Val Asn SerGly Arg Ser Ser Gly Ser Arg 1 5 10 7 6 PRT Diploptera punctata 7 Tyr ProGln Glu His Arg 1 5 8 14 PRT Diploptera punctata VARIANT (14) May be Tyror Phe 8 Ala Tyr Ser Tyr Val Ser Glu Tyr Lys Arg Leu Pro Val Xaa 1 5 109 5 PRT Diploptera punctata VARIANT (5) May be Tyr or Phe 9 Asp Gly ArgMet Xaa 1 5 10 9 PRT Diploptera punctata VARIANT (9) May be Tyr or Phe10 Ala Pro Ser Gly Ala Gln Arg Leu Xaa 1 5 11 5 PRT Diploptera punctataVARIANT (5) May be Tyr or Phe 11 Gly Gly Ser Leu Xaa 1 5 12 6 PRTDiploptera punctata VARIANT (6) May be Tyr or Phe 12 Gly Asp Gly Arg LeuXaa 1 5 13 12 PRT Diploptera punctata VARIANT (12) May be Tyr or Phe 13Pro Val Asn Ser Gly Arg Ser Ser Gly Ser Arg Xaa 1 5 10 14 7 PRTDiploptera punctata VARIANT (7) May be Tyr or Phe 14 Tyr Pro Gln Glu HisArg Xaa 1 5

We claim:
 1. A compound of the formula R-X₁-Phe-Gly-X₂-NH₂ wherein X₁ isselected from the group consisting of Asn, Asp, Gly, Ser, and Ala, andX₂ is selected from the group consisting of Leu and Ile, and R isR₁-X₄-R₂-X₃- wherein (i) X₄ is Arg, (ii) R₁ is a hydrophobic moietyselected from the group consisting of a 9-fluoreneacetic acyl group, a6-phenyl hexanoic acyl group, and a 9-phenyl nonanoic acyl group, saidhydrophobic moiety being effective to render said compound amphiphilic,(iii) R₂ is selected from the group consisting of a bond, an amino acid,and a polypeptide group, said polypeptide group comprising all or aportion of an allatostatin neuropeptide which is naturally contiguous tothe C terminal pentapeptide X₃-X₁-Phe-Gly-X₂-NH₂, and which saidpolypeptide group is sufficiently small as to retain the hydrophobicityof said compound introduced by said hydrophobic moiety, and (iv) X₃ isselected from the group consisting of Tyr, Phe, and carboranyl alanine.2. The compound of claim 1 wherein R₂ is a bond.
 3. The compound ofclaim 1 wherein R₂ is selected from the group consisting of: Leu-,Ala-Tyr-Ser-Tyr-Val-Ser-Glu-Tyr-Lys-Arg-Leu-Pro-Val- (SEQ ID No. 1),Ser-Lys-Met-, Asp-Gly-Arg-Met- (SEQ ID No. 2), Asp-Arg-Leu-,Ala-Arg-Pro-, Ala-Pro-Ser-Gly-Ala-Gln-Arg-Leu- (SEQ ID No. 3),Gly-Gly-Ser-Leu- (SEQ ID No. 4), Gly-Asp-Gly-Arg-Leu- (SEQ ID No. 5),Pro-Val-Asn-Ser-Gly-Arg-Ser-Ser-Gly-Ser-Arg- (SEQ ID No. 6),Tyr-Pro-Gln-Glu-His-Arg- (SEQ ID No. 7), and Pro-.
 4. A compositioncomprising the compound of claim 1 and an inert carrier.
 5. Thecomposition of claim 4 wherein said carrier is water.
 6. A compound ofthe formula R-X₁-Phe-Gly-X₂-NH₂ wherein X₁ is selected from the groupconsisting of Asn, Asp, Gly, Ser, and Ala, and X₂ is selected from thegroup consisting of Leu and Ile, and R is R₁-X₄-R₂-X₃- wherein (i) X₄ isselected from the group consisting of a bond and Arg, (ii) R₁ is ahydrophobic moiety selected from the group consisting of a9-fluoreneacetic acyl group, a 6-phenyl hexanoic acyl group, and a9-phenyl nonanoic acyl group, said hydrophobic moiety being effective torender said compound amphiphilic, (iii) R₂ is selected from the groupconsisting of a bond, an amino acid, and a polypeptide group, saidpolypeptide group comprising all or a portion of an allatostatinneuropeptide which is naturally contiguous to the C terminalpentapeptide X₃-X₁-Phe-Gly-X₂-NH₂, and which said polypeptide group issufficiently small as to retain the hydrophobicity of said compoundintroduced by said hydrophobic moiety, and (iv) X₃ is selected from thegroup consisting of Phe and carboranyl alanine.
 7. A compound of theformula R-X₁-Phe-Gly-X₂-NH₂ wherein X₁ is selected from the groupconsisting of Asn, Asp, Gly, Ser, and Ala, and X₂ is selected from thegroup consisting of Leu and Ile, and R is R₁-X₄-R₂-X₃- wherein (i) X₄ isselected from the group consisting of a bond and Arg, (ii) R₁ is ahydrophobic moiety selected from the group consisting of a9-fluoreneacetic acyl group, a 6-phenyl hexanoic acyl group, and a9-phenyl nonanoic acyl group, said hydrophobic moiety being effective torender said compound amphiphilic, (iii) R₂ is selected from the groupconsisting of Leu-, Ala-Tyr-Ser-Tyr-Val-Ser-Glu-Tyr-Lys-Arg-Leu-Pro-Val-SEQ ID No. 1), Ser-Lys-Met-, Asp-Gly-Arg-Met- (SEQ ID No. 2),Asp-Arg-Leu-, Ala-Arg-Pro-, Ala-Pro-Ser-Gly-Ala-Gln-Arg-Leu- (SEQ ID No.3), Gly-Asp-Gly-Arg-Leu- (SEQ ID No. 5),Pro-Val-Asn-Ser-Gly-Arg-Ser-Ser-Gly-Ser-Arg- (SEQ ID No. 6),Tyr-Pro-Gln-Glu-His-Arg- (SEQ ID No. 7), and Pro-, and (iv) X₃ isselected from the group consisting of Tyr, Phe, and carboranyl alanine.